In addition to direct human consumption, vegetable oil has added value for livestock feed, due to its higher energy density and is also increasingly used as a primary source for biodiesel production, particularly in Europe. Vegetable oils with high oleic acid (a monounsaturated fatty acid), and/or low levels of saturate fatty acids, provide considerable health and cooking benefits when compared to saturated and polyunsaturated fatty acids. Kinney et al. (2002) Biochem. Soc. Trans. 30:1099-103; White and Weber (2003) “Lipids of the kernel,” in Corn: Chemistry and Technology 2nd Ed., Vol. 10, Eds. White & Johnson, American Association of Cereal Chemists, Inc., St. Paul, Minn., pp. 355-95.
Though not a typical oil crop, high-oil (HO) corn has attracted considerable attention because corn oil offers high nutritional value for human consumption, and corn meal forms a large proportion of world's animal feed stock. Weber (2003) “Lipids of the kernel,” In Corn: Chemistry and Technology 2nd Ed., Vol. 10, Eds. White & Johnson, American Association of Cereal Chemists, Inc., St. Paul, Minn., pp. 11-349; Shen et al. (2010) Plant Physiol. 153:980-7. The Illinois High-Oil (IHO) population (Moose et al. (2004) Trends Plant Sci. 9:358-64) and the Alexho Single-Kernel (ASK) synthetic population (Lambert et al. (2004) “Single kernel selection for increased grain oil in maize synthetics and high-oil hybrid development,” in Plant Breeding Reviews Part 1, Vol. 1, Ed. Janick, John Wiley & Sons, Inc., Hoboken, N.J., pp. 153-75) are examples of high-oil maize developed by breeders through recurrent selection. Seed oil content in these populations has reached as high as 22%, and oleic acid contents in both populations are also elevated. Poneleit and Alexander (1965) Science 147:1585-6; Zheng et al. (2008) Nat. Genet. 40:367-72. Maize varieties with very high oleic acid and/or low saturate fatty acids have also been reported. U.S. Pat. Nos. 6,770,801 and 6,914,176. However, large scale commercial HO maize hybrids have not been released due to significant reductions in grain yield and other undesirable agronomic traits associated with HO germplasm. Nonetheless, these high-oil lines have provided valuable materials for QTL mapping and gene discovery, because long-term selection has accumulated uncommon alleles for oil and fatty acid composition. Recent studies have identified several major oil and fatty acid composition QTLs in these germplasm. Mangolin et al. (2004) Euphytica 137:251-9; Dudley et al. (2004) Crop Sci. 44:1419-28; Willmot et al. (2006) Maydica 51:187-99; Clark et al. (2006) Crop Sci. 46:807-19; Beló et al. (2008) Mol. Genet. Genomics 279:1-10; Zheng et al. (2008), supra; Wassom et al. (2008) Crop Sci. 48:243-52; Wassom et al. (2008) Crop Sci. 48:69-78; Yang et al. (2010) Theor. Appl. Genet. 120:665-78. Despite a good understanding of the plant oil and fatty acid biosynthetic pathways and a number of QTLs identified, very few genes underlying these QTLs, particularly those for oil QTLs, have been cloned in maize. Zheng et al. (2008), supra; Beló et al. (2008), supra. Most of this can be attributed to the fact that each QTL, even the major ones, only explains a small portion (10% or less) of phenotypic variation and is affected by the environment.
However, another significant reason that few genes underlying oil and fatty acid QTLs in high-oil maize have been identified is that conventional methods for oil measurement are inadequate for determination of oil phenotypes in maize. For example, methods for oil measurement with reasonable throughput are usually whole seed and concentration-based, whereas most of maize seed oil (85-90%) is located in the embryo and determined by the oil concentration of the embryo and the proportion of the seed occupied by the embryo. White and Weber (2003), supra. This discrepancy may sometimes produce misleading oil data because seed oil concentration is also significantly affected by seed and endosperm sizes. Further, the determination of the total oil content of plant tissues is typically based on the gravimetric extraction of oil from tissue using an organic solvent. This technique requires a relatively large amount of tissue to allow an accurate mass determination. Direct measurements using pulsed NMR may also be used, but this technique also requires a large amount of tissue to have sufficient liquid oil for detection.
Thus, despite a need for improved oil phenotype determination in plants (including maize) that has existed at least since the development of high-oil corn, and therefore for more accurate oil measurement techniques that can be conducted using relatively small samples, such techniques have not heretofore been achieved.